A growing body of evidence reveals that many diseases ranging from age-related macular degeneration, artherosclerosis, rheumatoid arthritis, to cancer are related to angiogenesis, the formation of new blood vessels (Folkman, 2001). Among these angiogenesis-dependent diseases, cancer is the most targeted disease (Brem, 1999; Ferrara and Alitalo, 1999; Keshet and Ben-Sasson, 1999; Carmeliet and Jain, 2000). There are tens of new therapeutic reagents under development based on the theory of antiangiogenesis. In the seminal publication by Folkman, the growth of tumors in both the primary and metastatic sites relies on angiogenesis to support both nutrients and oxygen to tumors (Folkman, 1971). In the following three decades, it has become increasingly convincing that angiogenesis plays a pivotal role in the malignant phenotype. New blood vessel formation has been demonstrated as a critical prognostic factor as well as a therapeutic target in many tumors.
The understanding that tumor growth and metastasis closely relate to the extent of angiogenesis has prompted research laboratories and pharmaceuticals to develop strategies to inhibit angiogenesis, thereby cutting off the blood supply to tumors (Brem, 1999; Ferrara and Alitalo, 1999; Keshet and Ben-Sasson, 1999; Kerbel, 2001; Risau, 1998; Klohs and Hamby, 1999; Rosen, 2000; Burke and DeNardo, 2001; Taraboletti and Margosio, 2001; Glaspy, 2002). Despite the promise of the scientific rationales and scores of experimental drugs being studied in clinical trials, researchers have yet to see significantly positive results from these studies, given the exciting anticancer effects that were demonstrated in the preclinical animal experiments.
Two of the most followed clinical studies involved two endogenous angiogenesis inhibitors, endostatin and angiostatin. These proteins have been shown to be cancer-angiogenesis specific and have no effects on normal blood vessel growth. They have been shown to inhibit cancer growth in animal studies without significant side effects and induction of drug resistance. (Boehm et al., 1997). However, the results from human cancer clinical trials did not match the stunning outcome from the preclinical test (Thomas et al., 2003; Herbst et al., 2002; Eder et al., 2002). Tumor responses in these trials are extremely rare. If there are tumor responses, the rate of the tumor regression is very slow. In some cases, it took more than one year for a patient to see a tumor regress more than 25%. So far, no rapid tumor shrinkage has been demonstrated in clinical trial using these angiogenesis inhibitors. Although tumor responses were not commonly demonstrated in these clinical studies, these endogenous angiogenesis inhibitors did show a very favorable safety profile.
As opposed to the tumor-specific angiogenesis seen in the animal model, tumor-specific blood vessels have been developed for a considerably longer period of time. Therefore, the blood vessels in human tumors are more mature than those in mice tumors. In some embodiments, it will require a longer time of angiogenesis inhibition for these endogenous inhibitors to block the blood flow to tumor to the extent that apoptosis of cancer cells are triggered. These angiogenesis inhibitors exert their function by inhibiting the growth of cancer cells instead of killing the cancer cells. The mechanism of their effect is so called “cytostatic” instead of “cytotoxic”. As opposed to cytotoxic reagents such as chemotherapy drugs, these cytostatic angiogenesis inhibitors can not efficiently attack well-established tumor blood vessels often seen in late stage tumor. Thus, these reagents so far did not demonstrate dramatic anticancer effect in clinical trials where most of the patients enrolled are in late stage and exhausted most of the available treatments
In contrast to the relatively non-toxic yet less potent anti-angiogenic proteins, described elsewhere herein, various potent therapeutic proteins or polypeptides, and the nucleic acids encoding them, have been used in attempts to treat cancers (not necessarily just kill cancer cells) or were suggested for such use. These include, for example, suicidal proteins, apoptosis-inducing proteins, cytokines, interleukines, TNF family proteins, and nucleic acids encoding them. Specific examples include: GM-CSF, Interferon Alpha, Interferon beta, Interferon gamma, Interleukin-1 Beta, Interleukin-2, Interleukin-4, Interleukin-5, Interleukin-6, Interleukin-8, Interleukin-10, Interleukin-12, Interleukin-13, Interleukin-14, Interleukin-16, Interleukin-18, Interleukin-23, Interleukin-24, Tumor Necrosis Factor SuperFamily member 14, Tumor Necrosis Factor SuperFamily member 13B, Tumor Necrosis Factor Alpha, Tumor Necrosis Factor SuperFamily member 12, Intercellular Adhesion Molecule-1, Lymphocyte Function-Associated antigen-3, Co-Stimulatory Molecule B7-1, Co-Stimulatory Molecule B7-2, FMS-related tyrosine kinase 3 ligand, CD40 Ligand, Surface antigen CD70, T-cell activation cell surface glycoprotein ligand, Co-Stimulatory Molecule OX-40 ligand, TNF-related activation-induced cytokine, Tumor Necrosis Factor SuperFamily member 11, TNF-related activation-induced cytokine, Tumor Necrosis Factor SuperFamily member 11, Cytosine deaminase, HSV Thymidine Kinase, Fas ligand, Caspase 3, TGF-α1, TGF-α2, TRAIL, Bax, Bak, Bik, Bok, Noxa, a Bcl-2 family protein, Granulysin (NKG5), Granzyme A, Granzyme B, and Perforin.
For example, IL2 and Interferon-α (Glaspy, 2002) have been used in the treatment for renal cell carcinoma and melanoma. However, significant systemic toxicity is usually seen in the cancer patients, thereby limiting the increase of dose and their clinical effects. IL12 has demonstrated potent and broad anticancer effects (Trinchieri, 2003). However, unacceptable side effects have manifested in some clinical trials (Leonard et al., 1997), which hamper its promise as an anticancer reagent.
To minimize the systemic side effects of cytokines, such as interleukin, as well as those therapeutic proteins listed in Table 1, many proteins have been used to target these otherwise considerably toxic therapeutic proteins to tumor-specific blood vessel. In addition, small molecules have been also utilized for tumor imaging while coupled to proteins specific for targeting tumor angiogenic blood vessels. Some of these approaches are summarized in Table 1.
TABLE 1Therapeutic/Targeting ToolDiagnostic AgentCommentsEndostatin99mTcSmall molecule 99mTC was used as imaging(Yang et al., 2002)molecule. The inventors used proteins as fusedmolecule, which could be utilized in gene therapywithout having to purify proteins.antibody fragmentIL-12The targeting antibody fragment is specific to onespecific to ED-Bof angiogenesis markers, ED-B domain ofdomain of fibronectinfibronectin. However, it does not possess(Halin et al, 2002)antiangiogenic activity.antibody fragmentIL-2Similar approach as (Hahn et al., 2002)specific to ED-Bdomain of fibronectin(Carnemolla et al.,2002)angiostatin-endostatinAngiostatin-Two antiangiogenic proteins were fused together(Scappaticci et al.,Endostatinand demonstrated better antiangiogenic effect than2001)single molecule. ThE new fusion protein stillcytostatic, but not cytotoxic.VEGFGeloninVEGF is specific to VEGF receptors, which are(Veenendaal et al.,diphtheriaexpressed abundantly in tumor vasculatures. It can2002; Arora et al.,(Veenendaaltrigger the angiogenic pathway. VEGF is not an1999; Hotz et al.,et al., 2002)antiangiogenic protein.2002)Toxin(Arora et al.,1999; Hotz et al.,2002)antibody B21-2 totruncated form ofThis approach again uses an antibody specific fortarget I-Ad, a markertissue factor (tTF)tumor blood vessel as targeting tool withoutof tumor specificintrinsic antiangiogenic property. tTF inducesblood vesselthrombosis, thereby blocking blood flow.(Huang et al., 1997)
Additional targeting strategies have involved the preparation of immunotoxins (Kreitman, 1999) by coupling antibodies specific to markers of tumor (CD20 of B-cell lymphoma, Her-2/neu of breast cancers, EGFR of colon, head and neck etc.) or tumor-specific blood vessels (ED-B domain of fibronectin, integrin αvβ3, VEGF receptors, etc.) to therapeutic reagents, such as interleukins, cytokines, gelonin, diphtheria toxin, radio-isotopes, etc. However, most of the immunotoxin strategies have yet to enjoy clinical success, except very few have been approved, such as Zevalin™ (ibritumomab tiuxetan) (IDEC Pharmaceuticals; San Diego, Calif.) and Baxxar (Corixa; Seattle, Wash.).
WO 99/16889 describes fusion proteins having an angiostatin amino acid sequence linked to a second moiety having different or complementary activity. In particular embodiments, the second moiety is selected from endostatin, human type I interferon, thrombospondin, interferon-inducible protein 10 (IP-10) and platelet factor 4. In other particular embodiments, the fusion proteins are used for anti-tumor treatment.
In view of the above, there is a need for compositions and methods that overcome the problems in the art and allow for the treatment of angiogenesis-dependent diseases.